The present invention relates, in general, to crystallization trays, and, in particular, to trays for forming diffraction-quality macromolecule crystals by vapor-diffusion techniques.
Supersaturated solutions of biological macromolecules (e.g., proteins, nucleic acids) under defined conditions form macromolecular crystals. Macromolecular crystals have been used in the biotechnology/pharmaceutical industry for many purposes. For example, three-dimensional structural models of macromolecule structures derived from X-ray crystallography are used to design new drugs and compositions in pharmaceutical and agricultural research; crystallization steps are utilized in purification/manufacturing processes of biotechnology-derived products; and crystalline complexes are used for controlled-release drug formulations and agricultural products, such as, for example, herbicides and insecticides.
One of the first and most important steps in the X-ray crystal structure determination of a target macromolecule is to grow suitably large, well-diffracting crystals of the macromolecule. Compared to the technological advances achieved in making, collecting, and analyzing X-ray diffraction data more rapid and automated, crystal growth has become a rate-limiting step in the structure determination process.
Vapor diffusion is the most widely used technique for crystallization in modern macromolecular X-ray crystallography. In this technique, a small volume of the macromolecule sample is mixed with an approximately equal volume of a crystallization solution to form a sample solution. A drop of the sample solution is sealed in a cell with a much larger reservoir volume of the crystallization solution. The drop is kept separate from the reservoir of crystallization solution either by hanging the drop from a cover slip (xe2x80x9changing dropxe2x80x9d technique) or by sitting the drop on a pedestal (xe2x80x9csitting dropxe2x80x9d technique) above the level of the crystallization solution in the reservoir. Over time, the crystallization drop and the equilibrating solution equilibrate via vapor diffusion of volatile chemical species. Supersaturating concentrations of the macromolecule are achieved, resulting in crystallization of the macromolecule sample in the drop.
Typically, several hundreds of experiments must be performed before conditions are found to produce high-quality crystals. Some of the conditions that are screened to determine the optimal conditions for crystal growth are pH, temperature, concentration of salts in the crystallization drop, concentration of the macromolecule to be crystallized, and concentration of the precipitating agent (of which there may be hundreds). Testing numerous combinations of conditions that affect crystal growth, by means of hundreds to thousands of crystallization experiments, eventually leads to the optimal conditions for crystal growth. Consequently, the ability to rapidly and easily generate many crystallization trials is important in determining the ideal conditions for crystallization.
Crystallization trays have been developed in the past in an effort to permit the efficient testing of numerous combinations of conditions that affect crystal growth. A problem with past trays is that the number of crystallization cells or chambers in a tray were too large and too few (e.g., 24 cells), causing the trays to be too large and/or limiting the number of crystallization experiments performed on a given tray. Another problem with past trays is that the crystallization cells did not have good viewing characteristics for viewing the crystallization process under a microscope or by using an imaging system. The cells often included solution reservoirs or sample receptacles that were too curved and/or were not clear, inhibiting viewing the crystallization process. A further problem with many past trays is that they are not sized to the standards set forth by the Society for Biomolecular Screening (SBS). Consequently, these trays can not be easily used with automated robotic equipment designed for trays meeting these standards. A still further problem with some past trays is that the trays are not formed as an integrated, single piece. For example, one tray in the past included multiple rows of crystallization cells that were removable from the tray base during use. The manufacture of multiple pieces made this tray expensive and susceptible to the cell rows accidentally dislodging from the tray base. Another problem with many trays in the past is that they are not appropriately designed for more general crystallography. Trays used for the hanging drop method have the disadvantage of not being easily used in automated processes because they require an automated mechanism for inverting the surface on which the drop is placed, such as a cover slip. But, many trays used for sitting drops are also not appropriate for maintaining the appropriately shaped drop. For example, in some crystallography trays, the floor of the sample solution receptacle is too flat and/or the floor includes sharp, angled corners. This may cause the sample solution to smear, cause a thin film to form, and/or cause the drop to migrate to the corner(s), inhibiting or preventing successful crystallization. If the drop migrates to a corner or corners, consistent visualization with a microscope is difficult because a consistent place may not exist in the cell for predicting where the sample is for visualization. In addition, commercially available trays do not provide for a simple way to seal individual cells without requiring the use of individual cover slips.
An aspect of the invention involves a crystallization cell including a reservoir adapted to receive an equilibrating solution, a shelf located adjacent to the reservoir and adapted for use as a temporary cryogenic holding area for a crystallized substance, and a sample drop receptacle carried by the shelf and adapted to receive a sample drop including a crystallizable substance.
Another aspect of the invention involves a crystallization tray. The crystallization tray includes a plurality of crystallization cells, each cell having a reservoir adapted to receive an equilibrating solution, a shelf located adjacent to the reservoir and adapted for use, for example, as a flat sample holding surface, and/or as a temporary cryogenic holding area for a crystallized substance, and a sample drop receptacle carried by the shelf and adapted to receive a sample drop including a crystallizable substance.
An additional aspect of the invention involves a crystallization tray including a base with a plurality of rectangular crystallization cells, a top wall, and a rectangular ridge extending upwardly from the top wall and delineating each cell. The ridge is adapted to support a cover for isolating each cell from ambient and from each other. The cover is preferably clear; preferred covers are self-adhesive, such as adhesive tape or plate sealer (available from, for example, 3M). The cover may also be one or a plurality of cover slips, covering either all of the crystallization cells, individual cells, or groups of cells such as rows or columns. Where the cover is not adhesive, the ridges of the tray could be greased with, for example, petroleum jelly, sealing medium, or grease, or gaskets could be used to attach the cover.
A still further aspect of the invention involves a method for forming macromolecular crystals. The method includes providing a macromolecule crystallization tray having a plurality of crystallization cells, each cell including a reservoir adapted to receive an equilibrating solution, a shelf located adjacent to the reservoir and adapted for use as a flat sample holding surface and/or as a temporary cryogenic holding area for a crystallized substance, and a sample drop receptacle carried by the shelf and adapted to receive a sample drop including a crystallizable substance; dispensing an equilibrating solution in the reservoirs, dispensing a plurality of macromolecular solution droplets in the sample drop receptacles, covering the cells with a cover; and crystallizing the crystallizable substance by vapor diffusion.
These and further objects and advantages will be apparent to those skilled in the art in connection with the drawing and the detailed description of the preferred embodiment set forth below.